Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Small interfering RNA (siRNA) is a naturally occurring dsRNA molecule, typically 21-23 nucleotides, processed from longer dsRNA (Bernstein et al. (2001) Nature 409:363-6) that selectively and persistently suppresses the expression of proteins through the catalytic, sequence-specific degradation of mRNA (Elbashir et al. (2001) Nature 411:494-8). As such, siRNA has tremendous potential in therapeutic applications where the suppression or absence of a protein or multiple proteins can produce a clinically beneficial result. Furthermore, many proteins identified as drug targets that cannot be inhibited by conventional low molecular weight drug candidates can potentially be inhibited by siRNA (Shen, Y. (2008) IDrugs 11:572-8).
siRNA can be administered directly as synthetic siRNA (typically 19 bp dsRNA with two 3′ nucleotide overhangs on each strand) that functions upon entry into the cytosol (Elbashir et al. (2001) Nature 411:494-8) or as pDNA that functions after entry into the nucleus and subsequent expression of short hairpin loops of RNA (shRNA) for cytosolic processing into siRNA (Brummelkamp et al. (2002) Science 296:550-3). siRNA is incorporated into an RNA-induced silencing complex (RISC) within the cytosol (Rand et al. (2004) Proc. Natl. Acad. Sci., 101:14385-9) where the sense strand is degraded to reveal the antisense strand (Matranga et al. (2005) Cell 123:607-20) and form an activated RISC. Activated RISC then facilitates the degradation of mRNA that is complementary to the loaded antisense strand (Ameres et al. (2007) Cell 130:101-12) and remains active (Hutvagner et al. (2002) Science 297:2056-60) for 3-7 days in dividing cells and for several weeks in non-dividing cells (Bartlett et al. (2006) Nucleic Acids Res., 34:322-33).
Although pDNA has the potential to deliver higher and more sustained dosages of siRNA, unpredictable and subsequently toxic dosages in vivo have been reported (Grimm et al. (2006) Nature 441:537-41). Furthermore, synthetic siRNA, unlike pDNA, does not need to enter and rely on the host nucleus for subsequent function. Thus, from a pharmaceutical perspective, synthetic siRNA is likely safer because dose can be tightly controlled and potentially more effective due to fewer intracellular barriers. Many clinical applications require the systemic administration of siRNA to achieve a therapeutic effect. The systemic administration of siRNA, however, is limited by several obstacles, including: (i) the extremely short plasma half-life of siRNA due to degradation by nuclease activity and renal clearance; (ii) low level cellular uptake of siRNA due to its large size (13 kDa) and negative charge; and (iii) the inability of siRNA to escape the endosomes/lysosomes into the cytosol, the site of activity (Whitehead et al. (2009) Nat. Rev. Drug. Discov., 8:129-38; Aigner, A. (2008) Curr. Pharm. Des., 14:3603-19; Howard, K. A. (2009) Adv. Drug Deliv. Rev., 61:710-20).
Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the systemic administration of siRNA (Aigner, A. (2008) Curr. Pharm. Des., 14:3603-19). The design of siRNA polyplexes requires balancing many requirements including: (i) protecting siRNA molecules against nuclease degradation in the plasma; (ii) increasing plasma half-life and providing a favorable distribution; (iii) promoting cellular uptake; (iv) facilitating endosome/lysosome escape of siRNA into the cytosol; (v) high biocompatibility/low toxicity; and (vi) absence of unwanted side effects (Whitehead et al. (2009) Nat. Rev. Drug. Discov., 8:129-38). The suppression of mRNA, however, is commonly low in many siRNA polyplexes. Thus, identifying designs and/or modifications that can increase mRNA suppression while satisfying the many other requirements for systemic administration is critical to the development of siRNA polyplexes.